1. Field of the Invention
The invention relates in general to purification of bioactive substances by biospecific adsorption. More specifically the invention relates to (a) a method of preparing a bioactive substance by adsorption on a mineral substrate in the aqueous phase, the substance being fixable to a given ligand by forming a specific stable reversible complex and the adsorbent carrier medium used being a finely divided mineral material modified by siloxane substituents carrying the ligands. The invention also relates (b) to a method of preparing the adsorbent substrate.
2. Description of the Prior Art
As is known, in order to purify biological substances (proteins, enzymes, heparin, hormones, lectins, antigens, antibodies and other polypeptides) by affinity chromatography, a chromatographic carrier material is selected and chemical molecules (ligands) having a strong specific affinity for the substance to be purified are grafted onto it. When the substance, to be purified is mixed with other substances from which it is to be separated, and placed in the presence of such a particular material, it becomes bonded to the ligand present on the material by a specific reaction (usually subsequently reversible), e.g. by forming complexes as in antigen-antibody reactions, whereas the other substances remain in solution. Subsequently the substance to be purified is salted out by placing the carrier material in a medium under conditions such that the complex dissociates and the desired substance and also the chromatographic carrier can be recovered and recycled in a new extraction process (see e.g. document U.S. Pat. No. 4,066,505). The salting out process is usually performed in a chromatographic column using an eluting solvent or solvent mixture.
The following are examples of carrier materials generally used for the aforementioned purposes: organic and mineral gels known commercially as Sephadex, Sepharose, Sephacryl, Spherosil, Ultogel, Affi-Gel, etc.
Usually these materials are made up of spherical or differently-shaped porous particles having sizes of the order of a few microns to a few hundred microns and pores measuring from 10 to 300 nm. The bonding agents for bonding a given ligand to the surface of the chromatographic carrier are molecules comprising a group capable of forming covalent bonds or other bonds with the ligand and also comprising a group for fixing the ligand (chemically or by adsorption) to the surface of the carrier. With regard to mineral carriers such as particles of silica (Spherosil) or porous glass (CPG glass), reactive silanes such as trialkoxy-alkyl silanes were the alkyl group comprises --OH, --CHO, --NH.sub.2, --NCO or oxirane which react with the ligands to be fixed and used.
For example K. Roy et al, in "Affinity Chromatography and Biological Recognition", ed. I. M. Chaiken et al, Academic Press 1983, page 257, describe the use of a silica gel carrier silanized with .gamma.-glycidyloxypropyl-trimethoxy-silane. P. O. Larsson et al in "some New Techniques Related to Affinity Chromatography", les Colloques de 1'INSERM: Chromatographie d'affinite et interactions molecularies, ed. J. M. Egli (1979), page 91, describe silanization of silica gel (10 .mu.m particles, 6 nm pores) by glyceropropyl trialkoxy silane (the alkyl substituent is a HOCH.sub.2 --CH.sub.2 OH--CH.sub.2 O--C.sub.3 H.sub.6 -group), the glycol group is converted to an aldehyde group by known methods and subsequently used to fix a bioactive ligand (e.g. an antibody) by a SCHIFF reaction with an amino group thereof followed by reduction of the N.dbd.C bond.
U.S. Pat. No. 3,652,761 describes the coupling of bioactive molecules (such as antigens or antibodies) to mineral carriers such as glass, silica gel, colloidal silica, bentonite, wollastonite, etc, by of alkylated alkoxysilanes where the alkyl group comprises amino, nitroso, carbonyl, carboxy, isocyanato, diazo, isothiocyanato, sulphydryl and halogenocarbonyl.
Document WO-A-82/02818 describes a method and device for separating immunoglobulins from milk by conveying the milk over a column filled with a bed of particles, such as a, porous glass silanized by the previously-mentioned conventional method and carrying monoclonal antibodies specific to one or the other of the immunoglobulins to be purified.
Other documents such as EP-A 56977 and EP-A 88818 discuss similar subjects.
The methods known hitherto and using the aforementioned techniques also have some disadvantages associated with the porous nature of the carriers used, the pore dimensions and the fact that the particles in the carriers must be substantially spherical in shape and have uniform dimensions to avoid channelling when placed in columns. Since most of the active surface of these particles is inside the pores and the pores often have dimensions of the same order of size as the molecules to be fixed, the molecules diffuse slowly inside the pores and, when salted out, have difficulty in diffusing towards the exterior of the particle. Also the substances (frequently macromolecules) to be purified only have low intrinsic diffusion speeds. Since the average distance to be travelled by the macro-molecules to reach the ligand in question is relatively large (a few tens of microns in the case of adsorbing particles) it is clear that the processes are slow and inefficient, whether for adsorption or desorption of substances to be purified. Small pores are not accessible to large molecules and ligands present in these pores will therefore be incapable of fixing the substance to be purified.
For the reasons given hereinbefore, the use of porous biospecific adsorbents have numerous disadvantages, mainly their low fixation capacity and the slowness of the adsorption and desorption steps. With regard to the fixation capacity, it is stated in document U.S. Pat. No. 3,652,761 that if the grafting rate is 14 to 18 mg of antigen (ligand) per gram of porous glass, only about 5 mg of antibody (human gamma-globulin) is fixed per g of carrier. Furthermore, the cost of porous beads of uniform dimension is very high and incompatible with large-scale industrial use.